The present invention relates to the field of somatic embryogenesis and, in particular, to methods of manipulating the maturation of somatic embryos within culture vessels.
Recently, somatic embryogenesis has gained attention as it offers a possible low-cost means for clonal reproduction of large numbers of plants of various species. The steps of somatic embryogenesis, including the initiation and proliferation of embryogenic cultures from explant tissues, have been documented in the art for many plants, including angiosperms and gymnosperms. Simply, the method of somatic embryogenesis involves the selection of an explant of a desired plant. The explant is removed from the parent plant tissue by excision and then subsequently cultured on at least one medium to produce a cell mass capable of further differentiation or development. The cell mass can be maintained and proliferated in the undifferentiated state indefinitely, or manipulated to stimulate differentiation into immature somatic embryo structures which can then be further cultured to form mature somatic embryos. Mature somatic embryos can be harvested and germinated immediately, or dried and then germinated, or dried and stored until required for germination.
Somatic embryos are known to be stimulated to develop and mature in culture if environmental stresses are imposed, such as heat, nutrient depletion, solute-based water stress or increased levels of the plant hormone abscisic acid ("ABA"), whether added exogenously or induced endogenously (see U.S. Pat. No. 5,238,835 to McKersie et al., the contents of which are incorporated herein by reference, said patent referred to hereinafter as the McKersie patent). The McKersie patent discloses the use of stress, including osmotic, nutrient, water and heat stresses among others, to trigger the endogenous production of ABA within somatic embryogenic cultures.
Due to the fact that somatic embryos develop without the surrounding nutritive tissues, i.e. megagametophytes in gymnosperm species and endosperm in angiosperm species, and protective seed coats normally present in zygotic seeds, research has focused on comparing the types and quantities of storage reserves (e.g. lipids, proteins, amino acids, monosaccharides and polysaccharides) produced in somatic embryos with those (average levels) in zygotic seeds of the same species, and on assessing their potential for improving the ease of handling, storage stability, and germination vigour of somatic embryos. Exogenous applications of ABA, and solutes such as polyethylene glycol ("PEG"--most commonly having a molecular weight of 4,000, but possibly ranging in molecular weight from 2,000 to 8,000) have been proposed as useful adjuncts for enhancing the levels of storage reserves in plant cells and in particular, somatic embryos. Specifically, it has been shown that ABA or PEG can be used to promote or otherwise enhance the maturation step of the somatic embryogenesis process with gymnosperms, e.g. conifers, and to reduce the occurrence of precocious germination during the maturation step (Roberts et al. 1990; Attree et al. 1991; Flinn et al. 1991; Carrier et al. 1997). The embryos which result from PEG and/or ABA facilitated maturation may be larger than their zygotic counterparts and may exhibit greater storage protein and lipid reserves (Flinn et al., 1991; 1995 U.S. Pat. No. 5,464,769 to Attree & Fowke, the contents of which are incorporated herein by reference, and said patent referred to herein as the Attree patent). Conifer somatic embryos produced on media containing PEG and having enhanced lipid levels and reduced moisture contents have been disclosed (the Attree patent). The use of ABA-amended media for the production of conifer somatic embryos with these same attributes have also been previously disclosed (Flinn et al. 1991: Carrier et al. 1997).
Accordingly, it is well known to increase the solute concentration in embryogenic culture media by the incorporation of permeating osmotica (i.e. sugars such as sucrose, mannitol or salts). However, there are problems inherent in these agents being absorbed by the symplast of the plant cells which leads to the development of atypical and poorly germinating embryo products. The alternative is to incorporate into the culture media, non-permeating high-molecular-weight compounds such as PEG or dextran (the Attree Patent). However, it has been recently disclosed that non-permeating high-molecular-weight solutes such as PEG and dextran do not reliably produce viable and useful embryos for all conifer species (Find et. al., 1997; Klimaszewska & Smith, 1997). It has also been disclosed that, contrary to common belief, small amounts of high molecular weight PEG (8000) enters the cell protoplast or alternatively, bind to the plasmalemma of Pinus taeda and sorghum callus cells when cultured on medium containing PEG (Newton et al., 1990). As well, concerns were raised about the adverse action of some unknown organic impurities in commercial PEG sources in the cellular metabolic processes (Plant and Federman, 1985).
A large group of patents held by the Weyerhaeuser Corporation discloses altering the osmotic potential of the medium during maturation of conifer somatic embryos using solutes. One representative patent is U.S. Pat. No. 5,563,061 which describes a multi-phase culturing process in which differently "tailored" media are used at each phase of somatic embryogenesis. During the second and third phases, the early stage embryos are grown for a defined time period on a culture medium containing a higher osmolality than that used in the induction phase. The osmotic potentials in the phase-two and phase-three media are altered by the incorporation of solutes such as sugars, PEG, sorbitol, myo-inositol, mannitol, and lactose.
Although the use of PEG or other similar non-permeating solutes discussed above as well as others known in the art, have been used successfully to mimic the chemical, hormonal, and environmental triggers of maturation in producing mature somatic embryos for some plant species including conifers, a large proportion of the embryos produced are atypical and not useful for germination and further propagation. Therefore, it is desirable to avoid the use of PEG and other similar non-permeating solutes for somatic embryogenesis with conifers generally, and with spruce and pine species in particular.